We have started working with primary rat peritoneal macrophages and we have some troubles detaching them from the surface of tissue plates. Could someone recommend me the best way to detach them without modifying macrophages phenotype? In order to purify and make sure that are mostly macrophages, we culture the peritoneal macrophages overnight before detaching them. Do you think it is too long and it is what makes more difficult the later detachment?
The following experiments might involve flow cytometry to study macrophages in general and then distinguishing between M1 and M2 macrophages, what markers can be used taking into the account that they are rat peritoneal macrophages. I have read about F4_80, CD64, CD80, CD163, CD14, CD86 and CD206. Unfortunately, some of them are not useful in rats. Does anyone know which surface markers works best when using flow cytometry?
Macrophages, regardless of species, are usually not easily released from plastic once they bind. The easiest thing to do, if you are not examining adherence molecules is with Trypsin/EDTA.
As for M1 and M2 markers... there is a paper that looked at markers in rat tissue. Article M1- and M2-macrophage Polarization in Rat Liver Cirrhosis In...
Hi Malen,
Accutase works best with cultured peritoneal/bone marrow macrophages (mouse, rat and human). You can pre-incubate macrophages with PBS for 5 min and then incubate cells with accutase at 37C. Usually 5-10 min is enough.
M1 and M2 states are related to functional characterization of macrophages. Instead of using surrogate cell surface markers you can directly measure protein expression of TNFa (e.g. NBP2-45331) , IL-1b ( e.g. NBP-1-19775), arginase (AF5868) or IL-10 (e.g. AF519) (All antibodies are from Bio-techne/Novus) using intracellular staining. Don't forget to stimulate cells with LPS (M1) or IL-4/TGFb (for M2).
I second the accutase method, but add that the cells will need to be pipetted (semi roughly) off in small wells or swirled in big plates. Make sure the PBS and accutase are prewarmed
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